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In vitro evaluation of xanthine oxidase inhibitory activity of Sonchus arvensis

leaves

Article  in  International Journal of Pharmacy and Pharmaceutical Sciences · January 2014

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 Academic Sciences International Journal of Pharmacy and Pharmaceutical Sciences

ISSN- 0975-1491 Vol 6 Issue 2, 2014

Research Article
IN VITRO EVALUATION OF XANTHINE OXIDASE INHIBITORY ACTIVITY OF SONCHUS
ARVENSIS LEAVES

RINI HENDRIANI*ab, ELIN YULINAH SUKANDARa, KUSNANDARANGGADIREDJAa, SUKRASNOa

aSchool of Pharmacy, Bandung Institute of Technology, Jalan Ganeca no. 10 Bandung, bFaculty ofpharmacy, Universitas Padjadjaran, Jalan
Ry Bandung-Sumedang, Jatinangor. Email: rhendriani@yahoo.com
Received: 01 Feb 2014, Revised and Accepted: 12 Mar 2014
ABSTRACT
Objective: This studyexaminedthe inhibiting effects of different extracts of S.arvensis leaves onthe activity of xanthine oxidase, an essential enzyme
for uric acid synthesis.
Methods: Activity test was conducted in vitro by measuring the activity of xanthine oxidase using UV spectrophotometry.
Results: The IC50 of ethanolic extract of S. arvensis leaves was 23.64 μg/mL. Meanwhile, the n-hexane, ethylacetateandwater fraction of the extract
had respective IC50 of 263.19, 16.20 and 141.80 μg/mL. When the leaves were extracted with ethyl acetate, the extract thus obtained showed an
IC50of 15.29 μg/mL.Allopurinol, as reference drug, had IC50 of 4.84 μg/mL.
Conclusion: Ethyl acetate extract of S. arvensis leaves potential to be developed as agent to treat hyperuricemia and gout.
Keywords: Sonchusarvensis, Gout, Xanthine oxidase, Inhibitory effect, In vitro.

INTRODUCTION authenticated atHerbarium Bandungense theSchool of Life

Gout is a painful inflammatory arthritis which can lead to decreasein
quality of life [1]. The disease occurs when body fluids are saturated
due to high levelof uric acid that eventually settles in the joints. Gout
could be overcome by lowering the levels of uric acid in the blood to
normal limits. Uricosurics and uricostatics are drugs commonly used
for this purpose.Uricosuricdrugsworkby inhibiting thereabsorption
ofuricacidinthe kidneytubulesin a competitivefashion, so that
theremoval of uric acidthrough the kidneyscan be improved.
Uricostatic drug inhibits the work xanthine oxidase enzyme that
convertshypoxanthine into xanthine and xanthine into uric acid,thus
reducing uric acid production. Uric acid is the final product of purine
metabolism in human body, that exits the body through urinary
excretion. This substance has very low solubility and tend to form
crystals. Uric acidaccumulated in the jointsis commonlyfoundinthe
form of monosodiumuriccrystals,thatinducesinflammatoty
reactions[2,3].A representative drug widely used to treat
hyperuricemia and gout is allopurinol. Allopurinol, which works by
preventing the formation of uric acid through the inhibition of the
enzyme xanthine oxidase, is particularly useful in dealing with
chronic gout characterized by tophi formed by deposits of uric acid
crystals in the joints, kidneys, and soft tissue. The tophaceous bodies
will eventually impaired renal function [4]. Apart from its efficacy,
allopurinol can cause hazardous side effects such as nephropathy,
allergic reactions and increase in toxicity of 6-mercaptopurine [5].
Furthermore, the drug could also inducehepatitisand allergic
reactions [6]. This fact warrantsthe search for new alternatives to
lower uric acid with minimal side effects. Many has now turned to
plant-derived substances as the source of medication. This research
was conducted in an attempt to develop substances with xanthine
oxidase inhibiting activity derived from the plant Sonchus arvensis.
This plant has been traditionally used in Indonesia as gout remedy
[7].In the previous studies the species of sonchus has been known
for its diverse activities and has been used in relieving disorders
such as hepatotoxicty [8] nephrotoxicity [9], cardiotoxicity [10],
asthma [11], and oxidative stress [12]. As to its effect on xanthine
oxidase, however, no in depth studieshas been conducted.
MATERIALS AND METHODS
Sciencesand Technology, Bandung Institute of Technology.
Plant Extraction
This study used the cold maceration method for extraction, with96%
ethanol and ethyl acetateas extracting solvents for the driedand
groud plant materials. The macerate was collected once everyday,
followed by soaking the residue with the same solvent system. This
procedure was repeated for three consecutive days. The macerate
thus accumulated was concentrated using rotary evaporator under
reduced pressure. This was followed by fractionation of the
ethanolic extract using n-hexane, ethylacetateandwater.
Xanthine oxidase inhibitory assay
Activity test was conducted in vitro by measuring the activity of the
enzyme xanthine oxidase using UV spectrophotometry
[6,13,14,15,16,17] with the slight modification. Xanthine oxidase
enzyme from bovine milk was prepared by dilution of the enzyme to
a final concentration 2 Units/mL. 1 mM xanthine substrate solution
was made by adding 5 drops of 1.0 M NaOH to increase the solubility
of xanthine. Ethanolic and ethyl acetate extracts and also fractions of
ethanol extract were dissolved in 1% dimethyl sulfoxide (DMSO)
and made the test concentration at 50, 100 and 200 mg/mL.
Allopurinolis usedas apositivecontrol.
Total volume of the assay mixture was 3.2 mLconsisting of 1 mL
sample test plant studied at various concentration, 1mL of 0.15 M
phosphate buffer (pH 7.8), 100 µL solution of the enzyme xanthine
oxidase. After preincubation of the test solution at 37 °C for 15 min, the
reaction was initiated by addition of 100 µL of xanthine substrate
solution and incubated at 37 °C for 30 min.
The reaction was stopped by adding 1 mL of 1N HCl. Spectrophotometer
absorbance at 295 nm, suggesting the formation of uric acid. Percent of
inhibition of xanthine oxidase activity of the test sample was determined
by measuring the absorbance of uric acid from the mixture without test
extracts (blank samples) compared with the absorbance of a mixture of
test extracts. IC50 values were obtained by linear regression analysis of a
plot a series of different sample concentrations against percent
inhibition.

Plant materials RESULTS

S. arvensisleaves, was collectedduring the period of December As shown in Table 1, Results of inhibitory test on xanthine oxidase
2012throughMarch 2013from thebotanicalgarden of Manoko in activity showed that the ethanolic extrac of S. arvensisleaves had IC50
Lembang, West Java, Indonesia.The plant materials were of 23.64 μg/mL, and the ethyl acetate extract gave an IC50 value of
 Rini et al.
Int J Pharm Pharm Sci, Vol 6, Issue 2, 501-503

15,29 μg/mL Meanwhile, the fractions of n-hexane, 263.19, 16.20 and 141.80 μg/mL. Table 1 further shows that, used as
ethylacetateandwater of ethanolic extract had the respective IC 50 of reference drug, allopurinol had IC50 value of 4.84 μg/mL.

Table 1: It shows that xanthine oxidase inhibitory ictivity of Sonchus Arvensisleaves. The leaves were firstly extracted with ethanol and
ethyl acetate. The ethnolic extract was further fractionated with n-hexane, ethyl acetate and water. All extracts and fractions were tested
for their xanthine oxidase inhibitory activity using UV spectrophotometry
Plant material Concentration (μg/mL) IC50 p
50 100 200
Ethyl acetate extract 54.07 ± 14.65 72.25 ± 17.43 86.75 ± 18.51 15.29 0.855
Ethanolic extract 47.37 ± 7.08 82.18 ± 8.86 88.45 ± 14.65 23.64 0.932
Ethyl acetat fraction of Ethanolic extract 52.06 ± 14.79 72.80 ± 17.82 83.16 ± 17.91 16.20 0.778
n-Hexane fraction of Ethanolic extract 8.19 ± 1.71 15.46 ± 5.61 37.84 ± 10.97 263.19 0.007
Water fraction of Ethanolic extract 17.82 ± 7.59 28.20 ± 8.10 74.55 ± 12.64 141.80 0.061
Allopurinol 59.14 ± 3.26 71.38 ± 7.01 91.75 ± 11.99 4.84 -
p values were obtained from comparison of treatment groups to Allopurinol.
Results of phytochemical screening, as shown in Table 2, demonstrated that the drug contained flavonoid and steroids/triterpenoids.

Table 2: It showsresults of phytochemical screening of Sonchus arvensis leaves and its ethanolic and ethyl acetate extracts.
Chemical Class Ground Leaves Ethanolic extract Ethyl acetate extract
Alkaloids - - -
Flavonoids + + +
Steroids/Triterpenoids + + +
Saponins - - -

DISCUSSION
The present study was carried out to investigate the xathine oxidase
inhibitory activity of S. arvensis leaves extracts and fractions in
search for substances that might have potential as alternatives to
treat hyperucemia and gout.
Extraction was done on ground leaves of S. arvensis with cold
maceration followed by low pressure evaporation at mininal heating
to preserve active substances contained, which are mostly phenolic
and flavonoid such as kaemferol, quercetin, orientin, rutin,
hyperoside, catechin and myricetin [18]. Flavonoids have been
known to have blood uric acid lowering activity through inhibition
of xanthine oxidase. Kaemferol is a xanthine oxidase inhibitor
without any additional superoxide scavenging activity, while
quercetin and myricetin are inhibitors with additional superoxide
scavenging activity [19].
The synthesis of uric acid in the oxidative pathway occurs via the
conversion of hypoxanthine into xanthine, catalyzed by the eznymes
xanthine oxidase and guanase. This is followed by oxidation of
xanthine into uric acid, which is also catalyzed by xanthine oxidase.
Inhibition of xnthine oxidase is thus essential as pharmacological
intervention for hyperuricemia and gout [2].
Results showed that the ethyl acetate fraction gave the highest
inhibition among the fractions tested. However, ethyl acetate extract
obtained from direct extraction of the leaves was shown to be
superior in terms of activity.These findings indicated that more
bioactive components were extracted with direct extraction with
ethyl acetate. Many biological activites exerted by plant extracts
have been associated with flavonoid content, known for effective
reactive oxygen-scavenging activity [18]. As shown by the result of
phytochemical screening, the crude drug, ethanolic as well as ethyl
acetate extract showed positive results for flavonoid. Further study
is needed to investigate whether flavonoid is the one responsible for
the xanthine oxidase-inhibiting activity.
CONCLUSION
S. arvensis under investigation exhibit xanthine oxidase inhibitory
activity. Results of present study suggest that ethyl acetate extract of
S. arvensis leaves has the most potent activity in inhibiting xanthine
oxidase activity, indicating its potential to be developed as agent for
treating hyperucemia and gout.
ACKNOWLEDGEMENT
We gratefully acknowledge the financial assistance received from
Bandung Institute of Technology through research grant funding
scheme to carry out this study.
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